Gene therapy refers to the use of genetic sequences and their introduction into cells to alter the genetic makeup of the cells and thereby change the properties or functioning of those cells.
Gene therapy may be used, for example, to correct a genetic defect by providing to the cells a good copy of a gene that functions as desired, or to provide a gene that encodes an RNA or protein that inhibits an undesired cellular or pathogen activity.
Gene therapy may be aimed at any of a variety of diseases in which there is a genetic aspect. Of particular interest are diseases of the blood or immune systems since the hematopoietic cells are relatively easy to collect from a subject, allowing for ex vivo procedures to be used. These include hemoglobinopathies, defects of leukocyte production or function, immune deficiencies, lysosomal storage diseases and stem cell defects such as Fanconi's anemia, chronic granulomatous disease, Gaucher's disease, G6PD deficiency etc. Many of these disorders have been successfully treated by allogeneic bone marrow cell transplants (Parkman 1986). However, the requirement for immune suppression or the occurrence of immunologic effects such as graft rejection are a disadvantage of allogeneic bone marrow transplantation. Gene therapy of hematopoietic stem cells has been suggested as an alternative means of treating disease affecting the hematopoietic system in humans.
Despite early promise of success in gene therapy in humans, clinical success has been very difficult to achieve despite a massive effort in the last decade (Mountain, 2000). This is due at least in part to low efficiencies of gene transfer, an inability to modify enough cells, an inability to target appropriate cell types, and a lack of persistence of the desired effect in human subjects.
Gene therapy of human hematopoietic stem (HS) cells has proven to be difficult to carry out in practice (Kohn et al 1998, Halene and Kohn 2000, Kume et al 1999). In most trials in humans, the level of gene-containing peripheral blood leukocytes has been low and these have been short-lived, suggesting a failure to transduce reconstituting HS cells (Bordignon et al 1995, Kohn 1995, Kohn et al 1998, Dunbar et al 1995, Hoogerbrugge et al 1996). This is related in part to the relatively few HS and hematopoietic progenitor (HP) cells in the body (Bertolini et al 1998, Reis 1999) and the requirement that the cells be activated when using some murine retroviral vectors for transduction. This is related to the low level of amphotropic receptors in quiescent human HS cells (Bodine et al 1998). Most human HS cells are quiescent, are relatively slow to respond to stimulation (Hao et al 1996, Gothot et al 1998) and when induced to divide, tend to lose long term repopulating capacity (Traycoff et al 1998). Almost all gene therapy attempts in humans using HS cells have up to now suffered from these two basic problems: insufficient numbers of HS cells that are totipotent and capable of long term engraftment have been transduced in order to have a therapeutic effect, and, secondly, the transduced cells have not persisted to provide modified hematopoietic cells long term.
The most promising trial of gene therapy into human HP cells involved the transfer of a gene into children with X-linked severe combined immunodeficiency (SCID) which led to the reconstitution of an immune system with gene-containing T-lymphocytes (Cavazzana-Calvo et al 2000; Hacein-Bey-Abina et al 2002). That trial used CD34+ cells from bone marrow of pediatric patients (<12 months) and delivered more than 106 transduced cells per kg. The number of CD34+ cells (per kg weight) that can be isolated from children, particularly of low weight, is much higher than in adults. Thymopoiesis is also more active in children. Furthermore, this study is unusual in that thymopoiesis in the SCID-X1 context results only from CD34+ cells that contain the exogenous gene (Cavazzana-Calvo et al 2001). In some ways, this study is analogous to those where myeloablation is carried out in that the infused cells can fill the physiological space that is unoccupied in the SCID patient. Early studies with allogeneic bone marrow transplantation showed that HS cell engraftment was not sustained in patients that were not myeloablated, primarily because of the continued presence of the recipient HS cells (Parkman 1986). Therefore, conclusions drawn from prior engraftment studies using human HS cells in an ablative context cannot be simply transferred to the non-ablative system.
Other reports of human clinical trials for gene therapy of hematopoietic progenitor cells are less positive. Kohn et al 1999 reported results of a clinical trial using bone-marrow derived CD34+ cells from pediatric patients (8-17 yrs) transduced with a gene encoding an RRE decoy (RNA molecule) against HIV. This trial failed to achieve significant transduction and engraftment of progenitor cells. In another trial, patients with breast or ovarian cancer were treated with HP cells after transduction with a marker gene, after myeloablation, but only transient presence of marked cells was observed (Bagnis et al 2002). A clinical trial including three patients with Gaucher disease showed presence of the gene-containing vector in peripheral blood and bone marrow up to 3 months post-infusion but at very low levels (Dunbar et al 1998). In another example, a trial with five patients suffering from Chronic Granulomatous Disease (CGD) was carried out whereby the p47phox gene was introduced into CD34+ cells from peripheral blood. Although corrected neutrophils were found in peripheral blood during the first few months after infusion, they were undetectable at 6 months post-infusion (Malech et al 1997). Further, a trial to correct Fanconi Anemia where the complementation group C gene was inserted into CD34+ cells resulted in only transient detection of the gene in the patients post-infusion (Liu et al 1999).
The poor results in these trials may reflect the lack of a survival advantage of the corrected cells compared to the uncorrected cells, in contrast to the X-linked SCID case. Furthermore, in most of these examples, the manipulated cell populations were administered to patients with no or partial myeloablation, requiring that the transduced cells compete with the resident stem cells to engraft.
Other factors may be operating as well. HS cells can be reduced in number in patients with HIV infection (Marandin et al 1996), making it more difficult to obtain sufficient numbers of such cells. Moreover, HS cells of HIV-infected individuals are compromised in their replication and clonogenic capacities and show an enhanced propensity to apoptosis (Vignoli et al 1998, Zauli et al 1996). Mobilization of peripheral blood HP cells using granulocyte colony-stimulating factor (G-CSF) was demonstrated in HIV-infected individuals (Law et al 1999). Maximal mobilization was achieved after 4 days of G-CSF administration. The leukapheresis product contained approximately 3×106 CD34+ cells per kg. Law et al did not transduce the isolated CD34+ cells nor show that the isolated CD34+ cells were capable of engrafting a subject long term. They merely speculate that gene therapy of HP cells might provide a cure for HIV infection. They also comment that discussion of the number of stem cells required for gene therapy of AIDS is premature because of many uncertainties, including the engraftment potential of the genetically modified cells, the need for chemotherapy, the need for myeloablation or not, the requirement to establish a niche for the infused cells, and the unknown response of the microenvironment in the marrow of AIDS patients after infusion of cells.
The minimum number of CD34+ cells from peripheral blood required for efficient restoration of the hematopoietic system, particularly platelet recovery, in the context of myeloablation has been suggested to be 2.0×106 cells per kg of weight of a subject (Zimmerman 1995). However, the number required for efficient engraftment when not performing myeloablation was unknown prior to this invention. It was unknown whether a “niche” had to be established for the infused cells, or the effect of competing, resident cells in the marrow. As mentioned above, this was particularly true in the context of HIV infection.
Many studies have used model animal systems, particularly in mice, to improve the methods for transduction and increase engraftment. However, although murine HS cells can be efficiently transduced with retroviral vectors, efforts to translate findings from the murine system to applications for human HS cells have revealed major difficulties (Halene and Kohn 2000; Richter and Karlson 2001).
A further difficulty for therapeutic application of gene therapy is in scaling up procedures to obtain sufficient transduced cell numbers (Schilz et al, 2000). Schilz et al measured transduction efficiency and engraftment in a mouse model, but it is unclear how the conclusions might apply to human subjects.
Each of these factors is addressed by the present invention.